Compositions for anti-inflammatory, antioxidant effects and improved respiratory function by specific histone deacetylase inhibition

ABSTRACT

Compositions comprising LSF compositions and treatment regiments comprising administration of LSF containing compositions are disclosed. Compositions and/or regiments may optionally include the administration of vitamins, minerals, and anti-oxidants. Methods for using these compositions and treatment regimens for treating subjects for diseases, including diseases associated with inflammation and/or oxidative stress, are provided. Various methods for use of the LSF compositions for inhibition of histone deacetylases (HDACs) in various cells, tissues, and/or conditions are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 14/793,839, filedJul. 8, 2015, which claims priority to Provisional Application U.S. Ser.No. 62/022,433 filed on Jul. 9, 2014, which are herein incorporated byreference in their entirety.

FIELD OF THE INVENTION

The invention relates to compositions comprising L-sulforaphane (LSF)and to treatment regiments comprising L-sulforaphane (LSF) compositions.Compositions and/or regiments may optionally include the administrationof vitamins, minerals, and/or anti-oxidants. Methods for using thesecompositions and treatment regimens for treating subjects for diseasesand conditions related to inflammation and/or oxidative stress, such aspulmonary edema and exercise-induced pulmonary hemorrhage, are provided.The invention further relates to various methods for use of the LSFcompositions for inhibition of histone deacetylases.

BACKGROUND OF THE INVENTION

Pulmonary edema is a condition caused by excess fluid in the lungs. Thisfluid collects in the numerous air sacs in the lungs, making itdifficult to breathe. The most common cause of pulmonary edema is heartproblems, but fluid can accumulate for other reasons, includingpneumonia, exposure to certain toxins and medications, and exercising orliving at high elevations.

Pulmonary edema that develops suddenly (acute) is a medical emergencyrequiring immediate care, and can sometimes prove fatal. Treatment forpulmonary edema varies depending on the cause, but generally includessupplemental oxygen and medications, and may require both acutetreatments along with ambulatory treatment for the underlying problem.

Oxidative stress and inflammatory responses are key features ofpulmonary edema and exercise-induced pulmonary hemorrhage (EIPH).Neutrophils and hemosiderophages (macrophages that have ingested anddigested red blood cells) are present in high numbers in the lungs ofanimals suffering from EIPH, indicating an influx of inflammatory cells.Similarly, hypoxia has been highly implicated

Pulmonary edema is of particular concern in elite athletes. For example,EIPH is an endemic production disease form of pulmonary edema of racingand other high-intensity exercise horses, which occurs when blood entersthe air passages of a horse's lung, which may lead to the impairment oflung function. EIPH or “bleeding” has been a recognized condition inracing horses for at least three hundred years, and has been reported tooccur in a variety of race horse breeds including racing Thoroughbreds(both flat racing and steeple chasing or jump racing), American QuarterHorses (incidence of 50-75%), Standardbreds (incidence of 40-60%),Arabians, and Appaloosas. EIPH has also been reported in eventers,jumpers, polo ponies, endurance horses, draft horses that pullcompetitively, and horses taking part in Western speed events such asreining, cutting and barrel racing. Virtually all horses that aresubjected to intense exercise bleed into the lungs, and these episodesof bleeding often commence as soon as these horses enter training,making EIPH a major welfare and economic concern to both veterinarians,and those involved in the racing and sport horse industries. Healingoccurs, but complete restoration of pulmonary function in the affectedarea often does not occur. Repeated episodes of intense exercise canresult in repeated episodes of pulmonary hemorrhage, and cumulativedamage to the affected lung tissue can occur such as e.g., fibrosisand/or scaring and consolidation of alveoli. These chronic changesoccur, particularly in the dorso-caudal lobes of the lung, and suchchanges can eventually curtail the performance of the horse.

Preventative/ameliorative/curative/restorative measures for EIPHaffected horses have also been sought for several hundred years. Formany years, the treatment of choice for prevention of EIPH in the racehorse has been pre-race treatment with the diuretic furosamide (Lasix®).However, the exact mechanism of action of furosamide in prevention ofEIPH is unknown, although many theories have been postulated over theyears, its effectiveness is in question, and its use in racing isillegal in all countries with the exceptions of the U.S. and Canada. Thetreatment of choice for EIPH, after the fact, is usually rest (mandatoryin many racing jurisdictions) and often in conjunction with antibioticsto prevent secondary bacterial infection and/or the use ofanti-inflammatory medication.

More recently, (following the research of West et al. J. Appl. Physiol.1993, 75: 1097-1109 related to the relationship of EIPH and increasedpulmonary artery pressure) attempts at treating EIPH via nitric oxideadministration have been tried, e.g., by Perry (U.S. Pat. No.5,765,548). Perry describes administration of nitric oxide throughcontinuous insufflation of the nitric oxide to the horse during theexercise period. Alternatively, the horse is treated with insufflationof nitric oxide prior to the exercise event and then is given anintramuscular injection of a phosphodiesterase inhibitor, e.g.,ZAPRINAST. The treatment during exercise as described by Perry is bothcumbersome and problematic for the racing animal and has never gainedwidespread acceptance. Likewise, systemic treatment of the racing animalwith phosphodiesterase inhibitors opens the door for unwanted sideeffects and requires regulatory scrutiny.

Histone deacetylases (HDACs) are a class of enzymes that remove acetylgroups (O═C—CH3) from an E-N-acetyl lysine amino acid on a histone,allowing the histones to wrap the DNA more tightly. Together with theacetylpolyamine amidohydrolases and the acetoin utilization proteins,the histone deacetylases form an ancient protein superfamily known asthe histone deacetylase superfamily. HDACs are classified in fourclasses depending on sequence homology to the yeast original enzymes anddomain organization. The Class I HDACs are HDAC1, HDAC2, HDAC3, andHDAC8. The Class IIA HDACs are HDAC4, HDACS, HDAC7, and HDAC9. The ClassIIB HDACs are HDAC6 and HDAC10. Class III HDACs include the sirtuinproteins (SIRT1-7). The HDAC11 is the Class IV HDAC. HDACs in Classes I,II, and IV (HDACs1-11) are metal-dependant HDACs. By modulating theacetylation status of histones, histone deacetylase inhibitors alter thetranscription of genes involved in cell growth, maturation, survival andapoptosis, among other processes. In addition to histones, HDACs havemany non-histone protein substrates which have a role in regulation ofgene expression, cell proliferation, cell migration, cell death, andangiogenesis.

The organosulfur compound L-sulforaphane (LSF) is obtained fromcruciferous vegetables (such as broccoli, Brussels sprouts or cabbages)when hydrolytic conversion of glucoraphanin to sulforaphane through theaction of physical damage to the plant occurs either by the action ofplant-derived myrosinase (intracellular broccoli thioglucosidase), or bythe microbiota of the human colon. Approximately, 60-80% ofglucoraphanin is converted to sulforaphane, with most broccoli varietiespossessing between 0.1 and 30 μmol/g of glucoraphanin.

LSF is known to have potent antioxidant effects by activation of theNrf2-ARE detoxification pathway. Nrf2 is a CNC (cap ‘n’ collar) bZIP(basic region leucine zipper) group of transcription factors which isbroadly expressed in a variety of tissues. Quiescent Nrf2 localizes inthe cytoplasm and is rapidly turned over through a specificubiquitin-26S proteasome pathway controlled by theKeap1/Cul3-independent ubiquitin ligase (E3). Nrf2 is activated inresponse to a range of oxidative and electrophilic stimuli includingROS, heavy metals and certain disease processes. Upon activation, Nrf2mediates antioxidant response by the induction of a broad range of genesincluding phase 2 enzymes, such as NAD(P)H:quinone oxidoreductase 1(NQO1) and heme oxygenase-1, and antioxidant proteins, such as SOD andcatalase. Both genetic and biochemical studies have implicated the Nrf2signaling pathway in the defense against a wide range of chemicaltoxicity, cancer and chronic diseases in which oxidative stress isinvolved. LSF has been shown to protect against oxidative stress andapoptosis by the induction of Nrf2-mediated antioxidant response.

Therefore, it is a primary object, feature, or advantage of the presentinvention to improve upon the state of the art.

It is a further object, feature, or advantage of the present inventionto provide methods of treating and/or preventing diseases associatedwith inflammation. In one aspect, the methods of treating and/orpreventing diseases associated with inflammation involve providing oradministering an effective amount of L-sulforaphane to a subject in needthereof. The L-sulforaphane may be combined with other components,including, for example, antioxidant or anti-inflammatory compounds. In aparticular embodiment, L-sulforaphane can be administered or provided incombination with one or more of hydroxytyrosol, oleuropein,N-acetylcysteine, L-proline, glycine, and taurine.

It is a further objective, feature or advantage of the present inventionto provide methods of treating and/or preventing pulmonary edema,including for example EIPH. In one aspect, the methods of treatingand/or preventing pulmonary edema involve providing or administering aneffective amount of L-sulforaphane to a subject in need thereof. TheL-sulforaphane may be combined with other components, including, forexample, antioxidant or anti-inflammatory compounds. In one embodiment,the methods involve providing or administering a nasal spray.

It is a further objective, feature or advantage of the present inventionto provide compositions and methods for inhibiting HDACs. In one aspectthe compositions and methods provide specific inhibition of Class IHDACs, and in particular embodiments specific inhibition of HDAC8.

It is a further objective, feature or advantage of the present inventionto provide compositions and methods for altering gene expression in acell, tissue, or subject, including by increasing lysine acetylation,and/or increasing or decreasing gene expression in cells or tissuescontacted with an LSF containing composition. These methods may be usedfor improving cell viability and/or treating or preventing oxidativestress in an individual or cell.

BRIEF SUMMARY OF THE INVENTION

The present invention provides compositions and methods for treating orpreventing pulmonary edema, including exercise-induced pulmonaryhemorrhage (EIPH). In one aspect, the invention encompasses compositionsand methods comprising L-sulforaphane (LSF) for treating or preventingpulmonary edema. LSF may be combined with other components, vitamins,minerals, and anti-oxidants, including one or more of hydroxytyrosol,oleuropein, N-acetylcysteine, L-proline, glycine, and taurine.

In another aspect, the invention provides methods of treating orpreventing conditions or diseases associated with inflammation oroxidative stress, comprising administering to a subject in need thereofa composition comprising LSF, an LSF derived and/or substitutedcompound, and/or an LSF analogue. In a preferred embodiment, the diseaseor condition is pulmonary edema or EIPH. In a more preferred embodiment,the subject is a human athlete or a horse.

In another aspect, the invention provides methods of inhibiting one ormore histone deacetylases (HDAC) in a cell comprising contacting saidcell with a composition comprising L-sulforaphane (LSF), an LSF derivedand/or substituted compound, and/or an LSF analogue. In a preferredembodiment, the HDAC is a Class I HDAC. In a more preferred embodimentthe HDAC is HDAC8.

In another aspect, the invention provides method for increasing ordecreasing gene expression in a cell, tissue, or subject, including byincreasing lysine acetylation of a histone polypeptide, using acomposition comprising L-sulforaphane (LSF), an LSF derived and/orsubstituted compound, and/or an LSF analogue. In a more particularaspect, the genes may be involved in type I (alpha/beta) and type II(gamma) interferon (IFN) signaling. In another aspect increasing ordecreasing of gene expression can be one or more of upregulation ofgeneral transcription factors (POL2, TAF1) and downregulation of STAT1,STAT2 and RAD21 targets.

In another aspect, the invention provides methods for improving cellviability and/or treating or preventing oxidative stress in anindividual or cell, comprising contacting said cell with a with acomposition comprising L-sulforaphane (LSF), an LSF derived and/orsubstituted compound, and/or an LSF analogue.

While multiple embodiments are disclosed, still other embodiments of thepresent invention will become apparent to those skilled in the art fromthe following detailed description, which shows and describesillustrative embodiments of the invention. Accordingly, the drawings anddetailed description are to be regarded as illustrative in nature andnot restrictive.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows binding of L-sulforaphane to histone deacetylase 8 relativeto the prototypical histone deacetylase inhibitor, Trichostatin A.

FIG. 2 shows inhibition of HDACs 1 and 8 by L-sulforaphane.

FIG. 3 (A-F) shows the effect of L-sulforaphane on cytokine andchemokine secretion from peripheral blood mononuclear cells (PBMC). PBMCwere stimulated in vitro with 1 μM Trichostatin A (TSA), 10 μMsuberoyanilide hydroxamic acid (SAHA), 10 mM sodium butyrate (NaB), 15μM LSF, 30 μM LSF or PBS (unstimulated), and production of (FIG. 3A)IL-6, (FIG. 3B) IL-1β, (FIG. 3C) IL-8, (FIG. 3D) IP-10 (FIG. 3E) MIP-1β,and (FIG. 3F) TNF-α were measured.

FIG. 4 (A-F) shows histological and immunofluorescence analyses of theeffect of L-sulforaphane on ovalbumin-induced allergic airways disease.(FIGS. 4A-4C) show H&E stained lung/bronchial tissue sections from micetreated with (FIG. 4A) saline (control), (FIG. 4B) vehicle control and(FIG. 4C) 5 mg/kg L-sulforaphane following challenge by Ovalbuminnebulisation. (FIGS. 4D-4F) shows immunofluorescence microscopy imagesof lung/bronchial tissue sections from mice treated with (FIG. 4D)saline (control), (FIG. 4E) vehicle control and (FIG. 4F) 5 mg/kgL-sulforaphane following challenge by Ovalbumin nebulization.

FIG. 5 (A-B) shows effects of L-sulforaphane on (FIG. 5A) mean airwaywall thickness and (FIG. 5B) epithelium thickness in a mouse model ofallergic airways disease.

FIG. 6 (A-B) shows L-sulforaphane (LSF) reduction of naphthalene-inducedepithelial denudation at 24 hours post-naphthalene injection withanalogous efficacy to dexamethasone. (FIG. 6A) Representativehematoxylin and eosin stained lung sections. (FIG. 6B) Quantitation ofhistological examination of stained lung sections. Corn Oil: CO;Naphthalene: NA; dexamethasone: DEX; L-sulforaphane: LSF.

FIG. 7 (A-B) L-sulforaphane (LSF) reduces naphthalene-induced thickeningof the lamina reticularis at 72 hours post-naphthalene injection withanalogous efficacy to dexamethasone. (FIG. 7A) Representative Mason'strichrome stained lung sections. (FIG. 7B) Quantitation of histologicalexamination of stained lung sections. Corn Oil: CO; Naphthalene: NA;dexamethasone: DEX; L-sulforaphane: LSF.

FIG. 8 shows L-Sulforaphane (LSF) attenuates doxorubicin-inducedaccumulation of γH2AX foci in H9c2 cells. Immunofluorescencevisualization of γH2AX foci (discrete foci in DAPI stained nuclei) inH9c2 cells pre-treated with 0, 10, 15 and 30 μM for 24 hours prior totreatment with doxorubicin.

FIG. 9 shows L-Sulforaphane (LSF) attenuates doxorubicin-inducedaccumulation of γH2AX foci in H9c2 cells. Quantification of γH2AX foci(discrete foci in DAPI stained nuclei) in H9c2 cells pre-treated with 0,10, 15 and 30 μM for 24 hours prior to treatment with doxorubicin.

FIG. 10 shows a multidimensional scaling (MDS) plot of gene expressionin PBMC from horses treated with LSF according to an exemplaryembodiment of the invention (LSF) and control untreated horses (C).

FIG. 11 shows a smear plot of gene expression changes in PBMC fromhorses treated with LSF according to an exemplary embodiment of theinvention, compared to control untreated horses. Grey points denotegenes with a false discovery rate (FDR) that is ≦0.05.

FIG. 12(A-B) shows Gene Set Enrichment Analysis (GSEA) plotsillustrating downregulation of genes involved in type I IFN signalling(FIG. 12A) and type II IFN signaling (FIG. 12B).

FIG. 13 (A-B) shows GSEA enrichment plots showing upregulation of genesco-regulated with MYST2, a histone acetyltransferase (FIG. 13A) anddownregulation of genes that have high expression in CD4 Tcells derivedfrom lupus patients (FIG. 13B).

FIG. 14 (A-B) shows GSEA enrichment plots illustrating downregulation ofSTAT1 target genes (FIG. 14A) and STAT2 target genes (FIG. 14B).

FIG. 15 (A-C) shows GSEA enrichment plots illustrating downregulation ofCD markers (FIG. 15A), immunoglobulins (FIG. 15B), and endogenousligands (FIG. 15C).

Various embodiments of the present invention will be described in detailwith reference to the drawings, wherein like reference numeralsrepresent like parts throughout the several views. Reference to variousembodiments does not limit the scope of the invention. Figuresrepresented herein are not limitations to the various embodimentsaccording to the invention and are presented for exemplary illustrationof the invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The embodiments of this invention are not limited to particularcompositions and methods of use thereof, which can vary and areunderstood by skilled artisans. It is further to be understood that allterminology used herein is for the purpose of describing particularembodiments only, and is not intended to be limiting in any manner orscope. For example, as used in this specification and the appendedclaims, the singular forms “a,” “an” and “the” can include pluralreferents unless the content clearly indicates otherwise. Further, allunits, prefixes, and symbols may be denoted in its SI accepted form.Numeric ranges recited within the specification are inclusive of thenumbers defining the range and include each integer within the definedrange.

So that the present invention may be more readily understood, certainterms are first defined. Unless defined otherwise, all technical andscientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art to which embodiments ofthe invention pertain. Many methods and materials similar, modified, orequivalent to those described herein can be used in the practice of theembodiments of the present invention without undue experimentation, thepreferred materials and methods are described herein. In describing andclaiming the embodiments of the present invention, the followingterminology will be used in accordance with the definitions set outbelow.

The term “about,” as used herein, refers to variation in the numericalquantity that can occur, for example, through typical measuring andliquid handling procedures used for making concentrates or use solutionsin the real world; through inadvertent error in these procedures;through differences in the manufacture, source, or purity of theingredients used to make the compositions or carry out the methods; andthe like. The term “about” also encompasses amounts that differ due todifferent equilibrium conditions for a composition resulting from aparticular initial mixture. Whether or not modified by the term “about”,the claims include equivalents to the quantities.

In the present invention, an “effective amount” or “therapeuticallyeffective amount” of a compound or of a composition of the presentinvention is that amount of such compound and/or composition that issufficient to affect beneficial or desired results as described herein.In terms of treatment of a mammal, e.g., a human patient, an “effectiveamount” is an amount sufficient to at least slow the progression orspread of disease, or render the disease susceptible to therapeutics orremediation.

The efficacy of the compositions in treating or preventing a particulardisease, disorder, or condition according to the present invention canbe evaluated both in vitro and in vivo. As used herein, the term“treating” refers to: (i) preventing a disease, disorder or conditionfrom occurring in a mammal, animal or human that may be predisposed tothe disease, disorder and/or condition but has not yet been diagnosed ashaving it; (ii) inhibiting the disease, disorder or condition, i.e.,arresting its development; and/or (iii) relieving the disease, disorderor condition, i.e., causing regression of the disease, disorder and/orcondition. For example, the compositions of the present invention may beused to prevent EIPH from occurring in racing horses (i.e. prior toexercise), to arrest the development of EIPH in racing horses (i.e.during exercise), and/or to relieve EIPH in horses (i.e. afterexercise). The efficacy of such compositions treatment may be measuredquantitatively or qualitatively to determine the presence/absence of thedisease, or its progression or regression using, in the example of EIPH,reduction in blood in the lungs, a reduction in inflammatoryinfiltration, a reduction or absence of other symptoms of EIPH, and/orno worsening in disease over a specified period of time or othersymptoms associated with the disease or clinical indications associatedwith the pathology of cancer development. In one aspect, this treatmentmay be accomplished by administering the compositions to a subject inneed thereof, for example by providing a nasal spray.

The term “weight percent,” “wt-%,” “percent by weight,” “% by weight,”and variations thereof, as used herein, refer to the concentration of asubstance as the weight of that substance divided by the total weight ofthe composition and multiplied by 100. It is understood that, as usedhere, “percent,” “%,” and the like are intended to be synonymous with“weight percent,” “wt-%,” etc.

As one skilled in the art shall appreciate, there are two distinctmechanisms for cell death. Apoptosis is the result of “normal” orprogrammed cell death. Through this physiological process cells areroutinely eliminated, giving balance to the proliferation of new cells.During apoptosis the outer membrane of the cell forms “bubbles” known asblebs. The content of the cells becomes incased in the blebs. The blebsseparate from the cell and are digested by nearby cells or macrophages.This orderly process greatly reduces toxicity to surrounding cells.

Inflammation refers to the process by which an organism attempts toremove injurious stimuli and to initiate the healing process,classically indicated by pain, heat, redness, swelling, and/or loss offunction. Inflammation may be either acute (the initial response of thebody to harmful stimuli primarily involving increased movement of plasmaand leukocytes from the blood into the injured tissues) or chronic. Theinflammatory response involves a cascade of biochemical events,implicating local vascular systems, the immune system, and various cellswithin the injured tissue. Inflammation may be detected or measured, forexample, by the presence of inflammatory cells, including white bloodcells such as neutrophils, monocytes/macrophages, B-cells, T-cells,NK-cells; myeloperoxidase (MPO) activity; and/or the presence ofinflammatory mediators, including cytokines and chemokines such asTNF-α, IL-1β, IL-6, IL-8, MIP-1β and IP-10.

The methods and compositions of the present invention may comprise,consist essentially of, or consist of the components and ingredients ofthe present invention as well as other ingredients described herein. Asused herein, “consisting essentially of” means that the methods andcompositions may include additional steps, components or ingredients,but only if the additional steps, components or ingredients do notmaterially alter the basic and novel characteristics of the claimedmethods and compositions.

Compositions

In an aspect of the invention the administration of LSF results inprevention or treatment of inflammation and/or oxidative stress. In oneembodiment, administration of LSF results in the prevention or treatmentof pulmonary edema, including for example EIPH. According to theinvention, the selective effects of LSF administration is mediated byspecific inhibition of histone deacetylases (HDACs), including HDAC8. Inan aspect, the LSF compositions are employed as a pre-treatment forsubjects that may develop pulmonary edema or EIPH, including humanathletes, individuals that will be at high altitude (elevation >2,500meters), and racing horses. The compositions according to the inventionprovide a biochemical mechanism by which cellular and/or systemiccharacteristics are regulated. The compositions and/or treatmentregimens according to the invention include LSF, and may include one ormore of hydroxytyrosol, oleuropein, N-acetylcysteine, L-proline,glycine, and taurine.

As referred to herein, LSF compositions include any LSF-based inhibitorof HDAC proteins. Suitable LSF-based inhibitor of the HDAC proteinsinclude, for example, LSF, a LSF derived compound, a LSF substitutedcompound, a LSF metabolite (originating from a prodrug), andcombinations of the same. A LSF composition may further include acarrier, diluent and/or other pharmaceutically acceptable deliveryagents or the like.

L-sulforaphane

The compositions according to the invention employ L-sulforaphane (LSF).L-sulforaphane (LSF; CAS Registry number [CAS 142825-10-3]), is alsoknown as (R)-1-Isothiocyanato-4-(methylsulfinyl)butane,4-Methylsulfinylbutyl isothiocyanate. LSF has the structure set outbelow:

For use in the composition of the present invention, LSF may be derivedfrom natural sources or prepared by chemical synthesis. For example, theLSF may be obtained as an extract of, or otherwise derived from, seeds,leaves, fruits, or other parts of cruciferous vegetables, and/orvegetation water of cruciferous vegetable production.

In addition to isolated, purified, derived and/or synthesized LSFcompositions, according to a further embodiment, a LSF derivative and/orsubstituted LSF, include for example sulforaphane-glutathione conjugatederivatives according to the following structure:

In addition, analogues of LSF can be employed for compositions andmethods of the present invention. Analogues may include compounds withthe following general formula:

Such analogues are understood to include any such compound wherein Rprovides a pharmaceutically acceptable salt, solvate, prodrug and/orisomer of LSF having the desired beneficial effect of treating orpreventing pulmonary edema, including EIPH. Such analogues can include,for example, 6-(Methylsulfinyl)hexyl isothiocyanate, D, L-sulforaphane,and (±)-4-methylsulfinyl-1-(S-methyldithiocarbamyl)-butane.

In a further embodiment, compounds derived from LSF (LSF derivatives),LSF substituted compounds, metabolites of LSF (its derivatives and/orsubstituted compounds), one or more mixtures thereof, or one or morecombinations thereof are employed for LSF compositions.

The term “prodrug” as understood by one skilled in the art refers tocompounds or derivatives that are converted in vivo to the compounds ofthe invention as a result of spontaneous chemical reaction(s), enzymecatalyzed chemical reaction(s), and/or metabolic chemical reaction(s).Examples of prodrugs include, but are not limited to, derivatives andmetabolites of the compounds of the formula set forth according to thepresent invention. These may include, for example, biohydrolyzablemoieties such as biohydrolyzable amides, biohydrolyzable esters,biohydrolyzable carbamates, biohydrolyzable carbonates, biohydrolyzableureides, and biohydrolyzable phosphate analogues. Further, prodrugsinclude compounds that can be oxidized, reduced, aminated, deaminated,esterified, deesterified, alkylated, dealkylated, acylated, deacylated,phosphorylated, dephosphorylated, or other functional group change orconversion involving forming or breaking chemical bonds on the prodrug,by either enzymatic action or by general acid or base solvolysis.Prodrugs can be prepared according to methods known to one skilled inthe art, such as those described by Burger “Medicinal Chemistry and DrugDiscovery 6th ed. (Donald J. Abraham ed., 2001, Wiley) and “Design andApplications of Prodrugs” (H. Bundgaard ed., 1985, Harwood AcademicPublishers). Without limiting the scope of the invention, any compoundthat is a prodrug of a compound of the formulas according to theinvention are included within the scope of the invention.

In a still further embodiment, LSF derivatives, substituted LSF and/orLSF analogues, including for example LSF acyl derivatives, substitutedhydroxyl groups and/or substituted compositions, are employed and havethe following general structure:

wherein R is selected from the group consisting of hydrogen, substitutedor unsubstituted alkyl, substituted or unsubstituted alkenyl,substituted or unsubstituted alkynyl, substituted or unsubstituted aryl,substituted or unsubstituted heterocyclyl, substituted or unsubstitutedacyl, ORa, SRa, SORa, SO2Ra, OSO2Ra, OSO3Ra, NO2, NHRa, N(Ra)2, ═N—Ra,N(Ra)CORa, N(CORa)2, N(Ra)SO2R′, N(Ra)C(═NRa)N(Ra)Ra, CN, halogen, CORa,COORa, OCORa, OCOORa, OCONHRa, OCON(Ra)2, CONHRa, CON(Ra)2, CON(Ra)ORa,CON(Ra)SO2Ra, PO(ORa)2, PO(ORa)Ra, PO(ORa)(N(Ra)Ra) and aminoacid esterhaving inhibitory efficacy against HDAC8 protein; and further whereinthe R group is selected from the group consisting of hydrogen,substituted or unsubstituted alkyl, substituted or unsubstitutedalkenyl, substituted or unsubstituted alkynyl, substituted orunsubstituted aryl, and substituted or unsubstituted heterocyclyl,substituted or unsubstituted acyl, and the like having inhibitoryefficacy against HDAC8 protein; and further wherein each of thesubstituted or unsubstituted alkyl, alkenyl, alkynyl, aryl,heterocyclyl, and/or acyl groups are C1-28 (including all rangestherein).

“Alkyl” refers to a straight or branched hydrocarbon chain radicalconsisting of carbon and hydrogen atoms, containing no unsaturation, andwhich is attached to the rest of the molecule by a single bond. Alkylgroups may include straight-chain alkyl groups (e.g., methyl, ethyl,propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, etc.), cyclicalkyl groups (or “cycloalkyl” or “alicyclic” or “carbocyclic” groups)(e.g., cyclopropyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl,etc.), branched-chain alkyl groups (e.g., isopropyl, tert-butyl,sec-butyl, isobutyl, etc.), and alkyl-substituted alkyl groups (e.g.,alkyl-substituted cycloalkyl groups and cycloalkyl-substituted alkylgroups). Unless otherwise specified, the term “alkyl” includes both“unsubstituted alkyls” and “substituted alkyls.” As used herein, theterm “substituted alkyls” refers to alkyl groups having substituentsreplacing one or more hydrogens on one or more carbons of thehydrocarbon backbone. Such substituents may include, for example,alkenyl, alkynyl, halogeno, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy,alkoxycarbonyloxy, aryloxy, aryloxycarbonyloxy, carboxylate,alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl,alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl,phosphate, phosphonato, phosphinato, cyano, amino (including alkylamino, dialkylamino, arylamino, diarylamino, and alkylarylamino),acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyland ureido), imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,sulfates, alkylsulfinyl, sulfonates, sulfamoyl, sulfonamido, nitro,trifluoromethyl, cyano, azido, heterocyclic, alkylaryl, or aromatic(including heteroaromatic) groups.

In some embodiments, substituted alkyls can include a heterocyclicgroup. As used herein, the term “heterocyclic group” includes closedring structures analogous to carbocyclic groups in which one or more ofthe carbon atoms in the ring is an element other than carbon, forexample, nitrogen, sulfur or oxygen. Heterocyclic groups may besaturated or unsaturated. Exemplary heterocyclic groups include, but arenot limited to, aziridine, ethylene oxide (epoxides, oxiranes), thiirane(episulfides), dioxirane, azetidine, oxetane, thietane, dioxetane,dithietane, dithiete, azolidine, pyrrolidine, pyrroline, oxolane,dihydrofuran, and furan.

Alkyl groups preferably have from 1 to about 22 carbon atoms. Methyl,ethyl, n-propyl, iso-propyl and butyl, including n-butyl, tert-butyl,sec-butyl and iso-butyl are particularly preferred alkyl groups. As usedherein, the term alkyl, unless otherwise stated, refers to both cyclicand noncyclic groups, although cyclic groups will comprise at leastthree carbon ring members, such as cyclopropyl or cyclohexyl. Alkylradicals may be optionally substituted by one or more substituents, suchas an aryl group, like in benzyl or phenethyl.

“Alkenyl” and “Alkynyl” refer to a straight or branched hydrocarbonchain radical consisting of carbon and hydrogen atoms, containing atleast one unsaturation (one carbon-carbon double or triple bondrespectively) and which is attached to the rest of the molecule by asingle bond. Alkenyl and alkynyl groups preferably have from 2 to about22 carbon atoms. The terms alkenyl and alkynyl as used herein refer toboth cyclic and noncyclic groups, although cyclic groups will compriseat least three carbon ring members. Alkenyl and alkenyl radicals may beoptionally substituted by one or more substituents.

“Aryl” refers to a radical derived from an aromatic hydrocarbon byremoval of a hydrogen atom from a ring carbon atom. Suitable aryl groupsin the present invention include single and multiple ring compounds,including multiple ring compounds that contain separate and/or fusedaryl groups. Typical aryl groups contain from 1 to 3 separated and/orfused rings and from 6 to about 22 carbon ring atoms. Aryl radicals maybe optionally substituted by one or more substituents. Speciallypreferred aryl groups include substituted or unsubstituted phenyl,substituted or unsubstituted naphthyl, substituted or unsubstitutedbiphenyl, substituted or unsubstituted phenanthryl and substituted orunsubstituted anthryl.

“Heterocyclyl” refers to a cyclic radical having as ring members atomsof at least two different elements. Suitable heterocyclyl radicalsinclude heteroaromatic and heteroalicyclic groups containing from 1 to 3separated and/or fused rings and from 5 to about 18 ring atoms.Preferably heteroaromatic and heteroalicyclic groups contain from 5 toabout 10 ring atoms. Heterocycles are described in: Katritzky, Alan R.,Rees, C. W., and Scriven, E. Comprehensive Heterocyclic Chemistry (1996)Pergamon Press; Paquette, Leo A.; Principles of Modern HeterocyclicChemistry W. A. Benjamin, New York, (1968), particularly Chapters 1, 3,4, 6, 7, and 9; “The Chemistry of Heterocyclic Compounds, A series ofMonographs” (John Wiley & Sons, New York, 1950 to present), inparticular Volumes 13, 14, 16, 19, and 28. Suitable heteroaromaticgroups in the compounds of the present invention contain one, two orthree heteroatoms selected from N, O or S atoms and include, e.g.,coumarinyl including 8-coumarinyl, quinolyl including 8-quinolyl,isoquinolyl, pyridyl, pyrazinyl, pyrazolyl, pyrimidinyl, furyl,pyrrolyl, thienyl, thiazolyl, isothiazolyl, triazolyl, tetrazolyl,isoxazolyl, oxazolyl, imidazolyl, indolyl, isoindolyl, indazolyl,indolizinyl, phthalazinyl, pteridinyl, purinyl, oxadiazolyl,thiadiazolyl, furazanyl, pyridazinyl, triazinyl, cinnolinyl,benzimidazolyl, benzofuranyl, benzofurazanyl, benzothienyl,benzothiazolyl, benzoxazolyl, quinazolinyl, quinoxalinyl,naphthyridinyl, and furopyridinyl. Suitable heteroalicyclic groups inthe compounds of the present invention contain one, two or threeheteroatoms selected from N, O or S atoms and include, e.g.,pyrrolidinyl, tetrahydrofuryl, dihydrofuryl, tetrahydrothienyl,tetrahydrothiopyranyl, piperidyl, morpholinyl, thiomorpholinyl,thioxanyl, piperazinyl, azetidinyl, oxetanyl, thietanyl,homopiperidinyl, oxepanyl, thiepanyl, oxazepinyl, diazepinyl,thiazepinyl, 1,2,3,6-tetrahydropyridyl, 2-pyrrolinyl, 3-pyrrolinyl,indolinyl, 2H-pyranyl, 4H-pyranyl, dioxanyl, 1,3-dioxolanyl,pyrazolinyl, dithianyl, dithiolanyl, dihydropyranyl, dihydrothienyl,pyrazolidinyl, imidazolinyl, imidazolidinyl, 3-azabicyclo[3.1.0]hexyl,3-azabicyclo[4.1.0]heptyl, 3H-indolyl, and quinolizinyl. Heterocylicradicals may be optionally substituted by one or more substituents.

In each of the aforementioned embodiments, the components of thecomposition of the present invention may optionally be present in theform of an ester or a physiologically and/or pharmaceutically acceptablesalt. Exemplary pharmaceutically acceptable salts refers to saltsprepared from pharmaceutically acceptable non-toxic acids, includinginorganic salts and organic salts. Suitable non-organic salts includeinorganic and organic acids such as acetic, benzenesulfonic, benzoic,camphorsulfonic, citric, ethenesulfonic, fumaric, gluconic, glutamic,hydrobromic, hydrochloric, isethionic, lactic, malic, maleic, mandelic,methanesulfonic, mucic, nitric, pamoic, pantothenic, phosphoric,succinic, sulfuric, tartaric acid, p-toluenesulfonic and otherpharmaceutically acceptable salts as provided in Stahl and Wermuth“Pharmaceutical Salts Properties, Selection, and Use”, 1st Ed,Wiley-VCH, 374 (2002).

In an aspect, the compositions according to the invention deliver atleast about 1 μM LSF, at least about 5 μM LSF, at least about 10 μM LSF,at least about 20 μM LSF, at least about 50 μM LSF, at least about 100μM LSF, or greater. In general, larger doses tend to produce greatereffects, with the preferred dosage also depending, at least in part,upon weight, metabolism, individual body chemistry, type of cancer orother condition being treated, and the like.

In an embodiment the dose of LSF administered to a person is about 0.01micrograms per kilogram of body weight to about 100 milligrams perkilogram of body weight. In addition, without being limited according tothe invention, all ranges recited are inclusive of the numbers definingthe range and include each integer within the defined range. In afurther aspect, the LSF is present at a level such that an effectiveamount for the reduction of inflammation and/or oxidative stress in thetarget cells or tissues results.

Depending upon the route of administration, greater doses of LSF may beadministered. For example, significantly lesser amounts of LSF may beabsorbed when the route of administration is inhaled (i.e. aerosol orspray) as compared to parenteral or other forms of systemicadministration. For inhaled delivery, therefore, the daily dose of LSFadministered by inhalation may be about 0.01 micrograms to about 1000micrograms per kilogram of body weight. By way of further example, inone embodiment, the daily dose of LSF administered to a subject byinhalation is about 1 to about 100 micrograms per kilogram of bodyweight. By way of further example, in one embodiment, the daily dose ofLSF administered to a subject by inhalation is about 5 to about 50micrograms per kilogram of body weight. By way of further example, inone embodiment, the daily dose of LSF administered to a subject byinhalation is about 10 micrograms to about 50 micrograms per kilogram ofbody weight.

For parenteral delivery the daily dose may be from about 0.01 to about100 micrograms per kilogram of body weight per day, twice a day, or morethan twice a day. In one embodiment, the daily dose of LSF parenterallyadministered to a person is about 0.1 to about 50 micrograms perkilogram of body weight per day. In another such embodiment, the dailydose of LSF parenterally administered to a person is about 0.1 to about10 microgram per kilogram of body weight.

Regardless of the route of administration of the LSF, the compositionsmay be administered in a single dose or multiple doses to achieve atarget daily dose. For example, for certain embodiments the LSF isprovided in a formulation that will provide a single daily dose.Alternatively, for such embodiments the LSF is provided in a formulationthat will provide, in two or more doses over the course of a day.

As one skilled in the art appreciates, greater amounts of LSF may beincluded in the dosage unit form when the intended route ofadministration is oral. For example, typical dosage forms for oraladministration include tablets, pills, capsules, gelcaps, caplets, andthe like. A single dose, therefore, may comprise a single tablet, pill,capsule, gelcap, caplet or the like, or two or more tablets, pills,capsules, gelcaps, caplets, and the like. In general, dosage forms fororal administration may contain 0.01 to 100 milligrams of LSF. Forexample, in one embodiment, the dosage unit form contains 1 to 50milligrams LSF.

The route of administration may affect the rate and extent of absorptionof LSF. Taking this into account, i.e., taking into account the fractionof an administered dose that is not absorbed or for whatever reason isnot systemically bioavailable to the subject, it is generally preferredthat the administered dose provide the subject with at least about 100but less than about 10,000, preferably less than about 6,000 TE ofsystemically bioavailable LSF per day. In general, it is preferred thatthe administered dose provide the subject with at least about 250 TE ofsystemically bioavailable LSF per day. In certain embodiments, it ispreferred that the administered dose provide the subject with at leastabout 500, at least about 750, at least about 1,000, or at least about5,000 TE of systemically bioavailable LSF per day.

Additional Functional Ingredients

The components of the treatment compositions according to the inventioncan further be combined with various functional components suitable foruse treating the particular cancer or other condition. Additionalfunctional ingredient components may include those that improve thehealth and/or viability of a patient and/or the cells of a patient.

In other embodiments, additional functional ingredients may be includedin the compositions. The functional ingredients provide desiredproperties and functionalities to the compositions. For the purpose ofthis application, the term “functional ingredient” includes a materialthat when combined with the LSF provides a beneficial property in aparticular use or treatment. Some particular examples of functionalmaterials are discussed in more detail below, although the particularmaterials discussed are given by way of example only, and that a broadvariety of other functional ingredients may be used.

In some embodiments, the compositions may include additional components,such as those that improves the health or viability of cells. In someaspects, such additional functional ingredients may include, for examplehydroxytyrosol, oleuropein, N-acetylcysteine, antioxidants, vitamins,minerals, and/or additional components. Such additional components, forexample, may include other antioxidants, vitamins, minerals, and/oramino acids. Non-limiting examples of other antioxidants includeascorbic acid (vitamin C) and its salts, ascorbyl esters of fatty acids,ascorbic acid derivatives (e.g., magnesium ascorbyl phosphate, sodiumascrobyl phosphate, and ascorbyl sorbate), EGCG, oleuropein, tocopherol(vitamin E), tocopherol sorbate, tocopherol acetate, other esters oftocopherol, tyrosol, butylated hydroxy benzoic acids and their salts,gallic acid and its alkyl esters such as propyl gallate, uric acid andits salts and alkyl esters, sorbic acid and its salts, lipoic acid,amines (e.g., N,N-diethylhydroxylamine and amino-guanidine), sulfhydrylcompounds (e.g., glutathione), dihydroxy fumaric acid and it salts,glycine pidolate, arginine pilolate, nordihydroguaiaretic acid,bioflavinoids, curcumin, lysine, methionine, proline, superoxidedismutase, resveratrol, and other polyphenols. In another embodiment,the composition comprises hydroxytyrosol, N-acetylcysteine, and one ormore of cystine, cystine derivatives, vitamin C, tannic acid, vitamin E,vitamin E derivatives, catechin, niacin, unsaturated fatty acids,vitamin P, vitamin Q, glutathione, isoflavones, guava, selenium,oleuropein or other polyphenol(s). In one embodiment, the compositioncomprises hydroxytyrosol, N-acetylcysteine and one or more of glycine,L-taurine, L-proline, niacinamide (vitamin B3), pyridoxine (vitamin B6),and methylsulfonylmethane.

In one embodiment, the composition contains non-amino acid additivessuch as aloe vera, oat extract, hyaluronic acid, betaglucan or likesubstance to provide glycosaminoglycans for extracellular matrixprotection. Vitamins may be additives, especially vitamins A/D3, all Bvitamins and all stable C vitamins. Omega 3 and 6 fatty acids will bebalanced with the greater percentage being 3. In one embodiment, thecomposition may contain other antioxidants, anti-inflammatory agents andtissue repair ingredients known to have wound healing benefits. Forexample, in one embodiment, the composition contains olive leaf extract,vitamin A/D3, Vitamin C, and essential fatty acids from olive oil,canola oil, safflower oil, borrage oil and sunflower oil. Alsopreferably, olive leaf extract is present in the composition of thepresent invention.

In one embodiment, the compositions include one or more of LSF,hydroxytyrosol, oleuropein, N-acetylcysteine, L-proline, glycine, andtaurine. In one embodiment, the composition contains N-acetylcysteineand hydroxytyrosol and the weight ratio of N-acetylcysteine tohydroxytyrosol to between 1:1 and 50:1, respectively. In one embodiment,the composition contains N-acetylcysteine and hydroxytyrosol and theweight ratio of N-acetylcysteine to hydroxytyrosol is between 10:1. and30:1, respectively. For example, in one such embodiment, the compositioncontains N-acetylcysteine and hydroxytyrosol and the weight ratio ofN-acetylcysteine to hydroxytyrosol is between 20:1 and 25:1,respectively.

In one embodiment, the composition contains glycine and hydroxytyrosoland the weight ratio of glycine to hydroxytyrosol to between 1:1 and50:1, respectively. In one embodiment, the composition contains glycineand hydroxytyrosol and the weight ratio of glycine to hydroxytyrosol isbetween 30:1 and 40:1, respectively. For example, in one suchembodiment, the composition contains glycine and hydroxytyrosol and theweight ratio of glycine to hydroxytyrosol is about 35:1, respectively.

In one embodiment, the composition contains L-taurine and hydroxytyrosoland the weight ratio of L-taurine to hydroxytyrosol to between 1:1 and50:1, respectively. In one embodiment, the composition containsL-taurine and hydroxytyrosol and the weight ratio of L-taurine tohydroxytyrosol is between 20:1 and 50:1, respectively. In oneembodiment, the composition contains L-taurine and hydroxytyrosol andthe weight ratio of L-taurine to hydroxytyrosol is between 30:1 and40:1, respectively. For example, in one such embodiment, the compositioncontains L-taurine and hydroxytyrosol and the weight ratio of L-taurineto hydroxytyrosol is about 35:1, respectively.

In one embodiment, the composition contains L-proline and hydroxytyrosoland the weight ratio of L-proline to hydroxytyrosol to between 1:1 and20:1, respectively. In one embodiment, the composition containsL-proline and hydroxytyrosol and the weight ratio of L-proline tohydroxytyrosol is between 1:1 and 10:1, respectively. In one embodiment,the composition contains L-proline and hydroxytyrosol and the weightratio of L-proline to hydroxytyrosol is between 1:1 and 5:1,respectively.

In one embodiment, the composition contains methylsulfonylmethane andhydroxytyrosol and the weight ratio of methylsulfonylmethane tohydroxytyrosol to between 1:1 and 30:1, respectively. In one embodiment,the composition contains methylsulfonylmethane and hydroxytyrosol andthe weight ratio of methylsulfonylmethane to hydroxytyrosol is between5:1 and 25:1, respectively. In one embodiment, the composition containsmethylsulfonylmethane and hydroxytyrosol and the weight ratio ofmethylsulfonylmethane to hydroxytyrosol is between 10:1 and 20:1,respectively.

In one embodiment, the composition contains niacinamide andhydroxytyrosol and the weight ratio of niacinamide to hydroxytyrosol tobetween 1:1 and 10:1, respectively. In one embodiment, the compositioncontains niacinamide and hydroxytyrosol and the weight ratio ofniacinamide to hydroxytyrosol is between 1:1 and 5:1, respectively. Inone embodiment, the composition contains niacinamide and hydroxytyrosoland the weight ratio of niacinamide to hydroxytyrosol is between 1:1 and2:1, respectively.

In one embodiment, the composition contains pyridoxine andhydroxytyrosol and the weight ratio of pyridoxine to hydroxytyrosol tobetween 1:1 and 10:1, respectively. In one embodiment, the compositioncontains pyridoxine and hydroxytyrosol and the weight ratio ofpyridoxine to hydroxytyrosol is between 1:1 and 5:1, respectively. Inone embodiment, the composition contains pyridoxine and hydroxytyrosoland the weight ratio of pyridoxine to hydroxytyrosol is between 1:1 and2:1, respectively.

In one preferred embodiment, the composition of the present inventioncontains hydroxytyrosol, N-acetylcysteine and optionally one or more ofglycine, L-taurine, L-proline, niacinamide (B3), pyridoxine (B6), andmethylsulfonylmethane. In one example of this embodiment, the weightratio N-acetylcysteine to hydroxytyrosol is between 1:1 and 50:1,respectively, the weight ratio glycine to hydroxytyrosol is between 1:1and 50:1, respectively, the weight ratio of L-taurine to hydroxytyrosolis between 1:1 and 50:1, respectively, the weight ratio of L-proline tohydroxytyrosol is between 1:1 and 20:1, respectively, the weight ratioof niacinamide to hydroxytyrosol is between 1:1 and 10:1, respectively,the weight ratio of pyridoxine to hydroxytyrosol is between 1:1 and10:1, and the weight ratio of methylsulfonylmethane to hydroxytyrosol isbetween 1:1 and 30:1. In another example of this embodiment, the weightratio N-acetylcysteine to hydroxytyrosol is between 10:1 and 30:1,respectively, the weight ratio glycine to hydroxytyrosol is between 30:1and 40:1, respectively, the weight ratio of L-taurine to hydroxytyrosolis between 20:1 and 50:1, respectively, the weight ratio of L-proline tohydroxytyrosol is between 1:1 and 10:1, respectively, the weight ratioof niacinamide to hydroxytyrosol is between 1:1 and 5:1, respectively,the weight ratio of pyridoxine to hydroxytyrosol is between 1:1 and 5:1,and the weight ratio of methylsulfonylmethane to hydroxytyrosol isbetween 10:1 and 30:1. In another example of this embodiment, the weightratio N-acetylcysteine to hydroxytyrosol is between 20:1 and 25:1,respectively, the weight ratio glycine to hydroxytyrosol is between 30:1and 40:1, respectively, the weight ratio of L-taurine to hydroxytyrosolis between 30:1 and 40:1, respectively, the weight ratio of L-proline tohydroxytyrosol is between 1:1 and 5:1, respectively, the weight ratio ofniacinamide to hydroxytyrosol is between 1:1 and 2:1, respectively, theweight ratio of pyridoxine to hydroxytyrosol is between 1:1 and 2:1, andthe weight ratio of methylsulfonylmethane to hydroxytyrosol is between10:1 and 20:1.

Composition Formulations

Compositions containing LSF may be formulated in any conventionalmanner. Proper formulation is dependent upon the route of administrationchosen. Suitable routes of administration include, but are not limitedto, oral, parenteral (e.g., intravenous, intraarterial, subcutaneous,rectal, subcutaneous, intramuscular, intraorbital, intracapsular,intraspinal, intraperitoneal, or intrasternal), topical (nasal,transdermal, intraocular), intravesical, intrathecal, enteral,pulmonary, intralymphatic, intracavital, vaginal, transurethral,intradermal, aural, intramammary, buccal, orthotopic, intratracheal,intralesional, percutaneous, endoscopical, transmucosal, sublingual andintestinal administration.

Pharmaceutically acceptable carriers for use in the compositions of thepresent invention are well known to those of ordinary skill in the artand are selected based upon a number of factors: LSF concentration andintended bioavailability; the disease, disorder or condition beingtreated with the composition; the subject, his or her age, size andgeneral condition; and the route of administration. Suitable carriersare readily determined by one of ordinary skill in the art (see, forexample, J. G. Nairn, in: Remington's Pharmaceutical Science (A.Gennaro, ed.), Mack Publishing Co., Easton, Pa., (1985), pp. 1492-1517,the contents of which are incorporated herein by reference).

In general, nasal routes of administration are preferred. Whenadministered nasally, these compositions are prepared according totechniques well known in the art of pharmaceutical formulation and maybe prepared as solutions in saline, employing benzyl alcohol or othersuitable preservatives, absorption promoters to enhance bioavailability,and/or other solubilizing or dispersing agents known in the art (see,for example, Ansel et al. (1999) Pharmaceutical Dosage Forms and DrugDelivery Systems (7th ed.).

The LSF containing compositions of the present invention may also bepreferably formulated for parenteral administration, e.g., formulatedfor injection via intravenous, intraarterial, subcutaneous, rectal,subcutaneous, intramuscular, intraorbital, intracapsular, intraspinal,intraperitoneal, or intrasternal routes. The compositions of theinvention for parenteral administration comprise an effective amount ofLSF in a pharmaceutically acceptable carrier. Dosage forms suitable forparenteral administration include solutions, suspensions, dispersions,emulsions or any other dosage form which can be administeredparenterally. Techniques and compositions for making parenteral dosageforms are known in the art.

Suitable carriers used in formulating liquid dosage forms for oral orparenteral administration include nonaqueous,pharmaceutically-acceptable polar solvents such as oils, alcohols,amides, esters, ethers, ketones, hydrocarbons and mixtures thereof, aswell as water, saline solutions, dextrose solutions (e.g., DW5),electrolyte solutions, or any other aqueous, pharmaceutically acceptableliquid.

Suitable nonaqueous, pharmaceutically-acceptable polar solvents include,but are not limited to, alcohols (e.g., .alpha-glycerol formal,.beta-glycerol formal, 1,3-butyleneglycol, aliphatic or aromaticalcohols having 2-30 carbon atoms such as methanol, ethanol, propanol,isopropanol, butanol, t-butanol, hexanol, octanol, amylene hydrate,benzyl alcohol, glycerin (glycerol), glycol, hexylene glycol,tetrahydrofurfuryl alcohol, lauryl alcohol, cetyl alcohol, or stearylalcohol, fatty acid esters of fatty alcohols such as polyalkyleneglycols (e.g., polypropylene glycol, polyethylene glycol), sorbitan,sucrose and cholesterol); amides (e.g., dimethylacetamide (DMA), benzylbenzoate DMA, dimethylformamide, N-(.beta.-hydroxyethyl)-lactamide,N,N-dimethylacetamide amides, 2-pyrrolidinone, 1-methyl-2-pyrrolidinone,or polyvinylpyrrolidone); esters (e.g., 1-methyl-2-pyrrolidinone,2-pyrrolidinone, acetate esters such as monoacetin, diacetin, andtriacetin, aliphatic or aromatic esters such as ethyl caprylate oroctanoate, alkyl oleate, benzyl benzoate, benzyl acetate,dimethylsulfoxide (DMSO), esters of glycerin such as mono, di, ortri-glyceryl citrates or tartrates, ethyl benzoate, ethyl acetate, ethylcarbonate, ethyl lactate, ethyl oleate, fatty acid esters of sorbitan,fatty acid derived PEG esters, glyceryl monostearate, glyceride esterssuch as mono, di, or tri-glycerides, fatty acid esters such as isopropylmyristrate, fatty acid derived PEG esters such as PEG-hydroxyoleate andPEG-hydroxystearate, N-methylpyrrolidinone, pluronic 60, polyoxyethylenesorbitol oleic polyesters such as poly(ethoxylated)30-60 sorbitolpoly(oleate)2-4, poly(oxyethylene)15-20 monooleate,poly(oxyethylene)15-20 mono 12-hydroxystearate, andpoly(oxyethylene)15-20 mono ricinoleate, polyoxyethylene sorbitan esterssuch as polyoxyethylene-sorbitan monooleate, polyoxyethylene-sorbitanmonopalmitate, polyoxyethylene-sorbitan monolaurate,polyoxyethylene-sorbitan monostearate, and Polysorbate® 20, 40, 60 or 80from ICI Americas, Wilmington, Del., polyvinylpyrrolidone, alkyleneoxymodified fatty acid esters such as polyoxyl 40 hydrogenated castor oiland polyoxyethylated castor oils (e.g., Cremophor® EL solution orCremophor® RH 40 solution), saccharide fatty acid esters (i.e., thecondensation product of a monosaccharide (e.g., pentoses such as ribose,ribulose, arabinose, xylose, lyxose and xylulose, hexoses such asglucose, fructose, galactose, mannose and sorbose, trioses, tetroses,heptoses, and octoses), disaccharide (e.g., sucrose, maltose, lactoseand trehalose) or oligosaccharide or mixture thereof with a C4-C22 fattyacid(s)(e.g., saturated fatty acids such as caprylic acid, capric acid,lauric acid, myristic acid, palmitic acid and stearic acid, andunsaturated fatty acids such as palmitoleic acid, oleic acid, elaidicacid, erucic acid and linoleic acid)), or steroidal esters); alkyl,aryl, or cyclic ethers having 2-30 carbon atoms (e.g., diethyl ether,tetrahydrofuran, dimethyl isosorbide, diethylene glycol monoethylether); glycofurol (tetrahydrofurfuryl alcohol polyethylene glycolether); ketones having 3-30 carbon atoms (e.g., acetone, methyl ethylketone, methyl isobutyl ketone); aliphatic, cycloaliphatic or aromatichydrocarbons having 4-30 carbon atoms (e.g., benzene, cyclohexane,dichloromethane, dioxolanes, hexane, n-decane, n-dodecane, n-hexane,sulfolane, tetramethylenesulfon, tetramethylenesulfoxide, toluene,dimethylsulfoxide (DMSO), or tetramethylenesulfoxide); oils of mineral,vegetable, animal, essential or synthetic origin (e.g., mineral oilssuch as aliphatic or wax-based hydrocarbons, aromatic hydrocarbons,mixed aliphatic and aromatic based hydrocarbons, and refined paraffinoil, vegetable oils such as linseed, tung, safflower, soybean, castor,cottonseed, groundnut, rapeseed, coconut, palm, olive, corn, corn germ,sesame, persic and peanut oil and glycerides such as mono-, di- ortriglycerides, animal oils such as fish, marine, sperm, cod-liver,haliver, squalene, squalane, and shark liver oil, oleic oils, andpolyoxyethylated castor oil); alkyl or aryl halides having 1-30 carbonatoms and optionally more than one halogen substituent; methylenechloride; monoethanolamine; petroleum benzin; trolamine; omega-3polyunsaturated fatty acids (e.g., alpha-linolenic acid,eicosapentaenoic acid, docosapentaenoic acid, or docosahexaenoic acid);polyglycol ester of 12-hydroxystearic acid and polyethylene glycol(Solutol® HS-15, from BASF, Ludwigshafen, Germany); polyoxyethyleneglycerol; sodium laurate; sodium oleate; or sorbitan monooleate.

A nasal preparation comprised of the composition described above cantake a variety of forms for administration in nasal drops, nasal spray,gel, ointment, cream, powder or suspension, using a dispenser or otherdevice as needed. A variety of dispensers and delivery vehicles areknown in the art, including single-dose ampoules, atomizers, nebulizers,pumps, nasal pads, nasal sponges, nasal capsules, and the like.

More generally, the preparation can take a solid, semi-solid, or liquidform. In the case of a solid form, the components may be mixed togetherby blending, tumble mixing, freeze-drying, solvent evaporation,co-grinding, spray-drying, and other techniques known in the art. Suchsolid state preparations preferably provide a dry, powdery compositionwith particles in the range of between about 20 to about 500 microns,more preferably from 50 to 250 microns, for administration intranasally.

A semi-solid preparation suitable for intranasal administration can takethe form of an aqueous or oil-based gel or ointment. For example, thecomponents described above can be mixed with microspheres of starch,gelatin, collagen, dextran, polylactide, polyglycolide, or other similarmaterials that are capable of forming hydrophilic gels. The microspherescan be loaded with drug, and upon administration form a gel that adheresto the nasal mucosa.

In a preferred embodiment, the nasal preparation is in liquid form,which can include an aqueous solution, an aqueous suspension, an oilsolution, an oil suspension, or an emulsion, depending on thephysicochemical properties of the composition components. The liquidpreparation is administered as a nasal spray or as nasal drops, usingdevices known in the art, including nebulizers capable of deliveringselected volumes of formulations as liquid-droplet aerosols. Forexample, a commercially available spray pump with a delivery volume of50 μL or 100 μL is available from, for example, Valois (Congers, N.Y.)with spray tips in adult size and pediatric size. In one embodiment, theLSF containing compositions are administered intranasally via an aerosolspray in a daily volume of between about 10 to 500 μL, more preferablybetween about 30 to about 200 μL.

The liquid preparation can be produced by known procedures. For example,an aqueous preparation for nasal administration can be produced bydissolving, suspending, or emulsifying the components in water, buffer,or other aqueous medium, or in a oleaginous base, such as apharmaceutically-acceptable oil like olive oil, lanoline, silicone oil,glycerine fatty acids, and the like.

It will be appreciated that excipients necessary for formulation,stability, and/or bioavailability can be included in the preparation.Exemplary excipients include sugars (glucose, sorbitol, mannitol,sucrose), uptake enhancers (chitosan), thickening agents and stabilityenhancers (celluloses, polyvinyl pyrrolidone, starch, etc.), buffers,preservatives, and/or acids and bases to adjust the pH, and the like.

Methods

The LSF containing compositions and/or regimens of the present inventionmay be used in methods for the treatment of subjects having a variety ofdiseases. In some embodiments, the LSF containing compositions and/orregimens of the present invention may be used for the treatment ofdiseases or conditions associated with inflammation or oxidative stress.In some embodiments, the LSF containing compositions and/or regimens ofthe present invention may be used for the treatment or prevention ofpulmonary edema, including exercise induced pulmonary hemorrhage (EIPH),or high-altitude pulmonary edema (HAPE).

In an embodiment, the treatment may be performed by administration of aspray or aerosol LSF containing compositions and/or regimens to thesubject in need thereof. The treatment may be performed in conjunctionwith administration of other beneficial compositions, for examplehydroxytyrosol-containing compositions according to U.S. Pat. No.8,765,794, which is incorporated herein in its entirety. The treatmentmay be performed by administration of components in any order and in anycombination. Further, the treatment may be performed by providingmultiple administrations of the compositions. One skilled in the artwill ascertain these variations in treatment regimens employing the LSFcompositions and/or regimens disclosed herein.

As referred to in the methods of administering LSF compositions, suchcompositions include any LSF-based inhibitor of HDAC proteins. SuitableLSF-based inhibitor of HDAC proteins include, for example, LSF, a LSFderived compound, a LSF substituted compound, a LSF metabolite(originating from a prodrug), and combinations of the same. A LSFcomposition may further include a chemotherapeutic agent, carrier,diluent and/or other pharmaceutically acceptable delivery agents or thelike.

The methods of the invention may be further applicable to otherconditions that are associated with inflammation or oxidative stress,such as for example, ankylosing spondylitis, multiple sclerosis, Crohn'sdisease, psoriasis, psoriatic arthritis, rheumatoid arthritis, andscleroderma.

The combination of LSF and optionally one or more of hydroxytyrosol,oleuropein, N-acetylcysteine, L-proline, glycine, and taurine accordingto methods of the invention results in at least additive effects,preferably synergistic effects. The combinational therapy according tothe invention results in a greater reduction of symptoms, including forexample greater reduction of fluid in the lungs, greater reduction ofhypoxia, and greater reduction of inflammation and/or inflammatorymediators, and/or other indicators of improved treatment for a regimendisclosed herein, in comparison to any compound alone.

Inhibition and Inactivation of Histone Deacetylases

The activity of HDACs is regulated on multiple levels includingprotein-protein interactions, post-translational modification byphosphorylation, acetylation, sumoylation and proteolysis, subcellularlocalization, and a variety of metabolic cofactors, including forexample zinc. Without being bound to any particular theory, compositionsand methods of the present invention may inhibit HDACs, by interferingwith or blocking interactions with substrates or other proteins. In oneaspect, LSF may occupy, mask, or otherwise block access to the catalyticsite of the HDAC.

In one aspect, the LSF containing compositions specifically inhibitClass I HDACs, and more particularly specifically inhibit HDAC8. Thus,LSF containing compositions of the present invention may be used forparticular HDAC inhibition.

LSF Interaction with Histone Deacetylase Enzymes (HDACs)

Without being limited to a particular theory, it is believed that LSFinteracts directly with HDACs, including specific interaction withHDAC8, in a manner that inhibits the deacetylase activity of the enzyme.In the alternative, LSF may block peptide ligand binding or alter theconformation of the ligand binding site of HDACs, including HDAC8, orinteracting with key amino acid residues that affect substrate bindingby HDACs, including HDAC8. The invention therefore embodies anyderivative of LSF or glucosinolates, or structural mimics or homologuesthereof, that exhibits binding and/or inhibition characteristics similarto LSF.

As demonstrated by this invention, LSF inhibits the enzymatic activityof HDAC8 (FIG. 2). Unlike other inhibitors of HDAC8, LSF is a naturallyoccurring compound, lacking the substantial toxic side effects of otherinhibitors. According to the invention, HDACs, including HDAC8, isinhibited by exposure to compositions comprising LSF, derivatives ofLSF, or structural mimics or homologues thereof. This inhibition ofHDACs prevents inflammatory responses, including the production ofinflammatory mediators. In addition, LSF is known to have potentantioxidant effects by activation of the Nrf2-ARE detoxificationpathway. Although not bound by this exemplary embodiment, LSF can beprovided in order to inhibit enzymatic activity, prevent associationwith co-factors or partner proteins, or otherwise inhibit HDACs, or toreduce oxidative stress in target cells or tissues, thereby treating orpreventing conditions associated with inflammation or oxidative stress.

It is understood that prevention or treatment of conditions associatedwith inflammation, including pulmonary edema and EIPH, by LSF, orderivatives/equivalents of LSF, can be by one or more of thesemechanisms.

Methods of Treating Diseases or Conditions Involving Inflammation and/orOxidative Stress

LSF reduces the production of inflammatory mediators, includingcytokines and chemokines. Methods according to the present invention mayinclude administration of LSF containing compositions to a subject inneed thereof in order to block or reduce an inflammatory response eithersystemically or at a specific location (i.e. in the airways and lungs).

In a more particular aspect, the methods of the present invention mayinvolve modulation of genes involved in type I (alpha/beta) and type II(gamma) interferon (IFN) signaling. Such modulation may be increasing ordecreasing the expression of one or more of the genes. Other methods ofthe present invention may involve modulation (i.e. increasing ordecreasing expression) of one or more genes due to upregulation ofgeneral transcription factors (POL2, TAF1) and/or downregulation ofSTAT1, STAT2 and RAD21 targets.

All publications and patent applications in this specification areindicative of the level of ordinary skill in the art to which thisinvention pertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated as incorporated by reference.

EXAMPLES

Embodiments of the present invention are further defined in thefollowing non-limiting Examples. It should be understood that theseExamples, while indicating certain embodiments of the invention, aregiven by way of illustration only. From the above discussion and theseExamples, one skilled in the art can ascertain the essentialcharacteristics of this invention, and without departing from the spiritand scope thereof, can make various changes and modifications of theembodiments of the invention to adapt it to various usages andconditions. Thus, various modifications of the embodiments of theinvention, in addition to those shown and described herein, will beapparent to those skilled in the art from the foregoing description.Such modifications are also intended to fall within the scope of theappended claims.

Example 1 L-Sulforaphane Inhibits Specific Metal-Dependent HistoneDeacetylase Enzymes Summary

L-sulforaphane exhibits specific affinity for binding particular histonedeacetylase enzymes (HDACs), and specific inhibition of HDAC8.

Methods

The binding of L-sulforaphane to the 11 metal dependent histonedeacetylase enzymes was performed using the epigenetic assay services.Experiments were performed in comparison to the prototypical histonedeacetylase inhibitor, Trichostatin A.

Results and Discussion:

The binding constant and hillslope for LSF in comparison to TrichostatinA is shown in Table 1. The findings indicate specific binding of LSF toHDACs 3, 6, 7 and 8 which is more subtle than Trichostatin A. Thebinding of LSF to HDAC8 is most pronounced and indicated in FIG. 1.

TABLE 1 Binding constants for LSF to metal-dependent histonedeacetylases. HDAC LSF Trichostatin A HDAC-1 HILLSLOPE −0.79 IC50 (M)3.41E−09 HDAC-2 HILLSLOPE −0.93 IC50 (M) 1.38E−08 HDAC-3 HILLSLOPE −0.20−0.49 IC50 (M) 0.02053 1.16E−08 HDAC-4 HILLSLOPE −0.24 IC50 (M) 1.24E−07HDAC-5 HILLSLOPE −0.77 IC50 (M) 7.61E−09 HDAC-6 HILLSLOPE −0.88 −1.16IC50 (M) 1.02E−03 1.39E−09 HDAC-7 HILLSLOPE −0.41 −0.49 IC50 (M)5.81E−03 4.17E−08 HDAC-8 HILLSLOPE −0.88 −0.64 IC50 (M) 8.27E−052.10E−07 HDAC-9 HILLSLOPE −0.45 IC50 (M) 2.49E−08 HDAC-10 HILLSLOPE−0.84 IC50 (M) 1.27E−08 HDAC-11 HILLSLOPE −0.59 IC50 (M) 1.05E−08The inhibition of HDAC1 and HDAC8 enzymatic activity by L-sulforaphanewere examined using the HDAC1 and HDAC8 inhibitor Screening Assay Kitsfrom Cayman Chemical, respectively, using the manufacturer'sinstructions. The results of these assays shown in FIG. 2, highlight thespecificity of L-sulforaphane for HDAC8 compared to HDAC1.

Example 2 Anti-Inflammatory and Antioxidant Effects of L-SulforaphaneSummary

L-sulforaphane reduces cytokine and chemokine release from peripheralblood mononuclear cells.

Methods

Cryopreserved peripheral blood mononuclear cells (PBMC) from healthyadult donors were rapidly thawed in a 37° C. water-bath untilapproximately 50% thawed and slowly re-suspended in 10 mL of RPMI-1640medium supplemented with 20 mmol/L HEPES (pH 7.4), 10% (v/v) fetalbovine serum, 2 mmol/L L-glutamine, and 20 μg/mL gentamicin(GIBCO-Invitrogen, USA). The PBMC suspension was centrifuged for 5minutes at 700 g, after which the supernatant was discarded and cellswere re-suspended in fresh RPMI-1640.

The levels of TNF-α, IL-1β, IL-6, IL-8, MIP-1β and IP-10 in supernatantsfrom PBMC samples stimulated in vitro with 1 μM Trichostatin A (TSA), 10μM suberoyanilide hydroxamic acid (SAHA), 10 mM sodium butyrate (NaB),15 μM LSF, 30 μM LSF or PBS (unstimulated) were measured using the MAPHuman cytokine/chemokine kit (Millipore, USA) as per manufacturer'sinstructions. The 96-well filter plate was pre-wet by adding 200 μL/wellof assay buffer and incubated on a shaker for 10 mins at RT. Assaybuffer was removed by vacuum and 254 of the standard and quality controlreagents were added in duplicate with a six-point standard curveprepared using the human cytokine/chemokine standard reagent using 1:5serial dilutions in the range 10,000 pg/mL-3.2 pg/mL and PBS (−) aloneas the background. Undiluted supernatants were added in duplicate (25μL/well) followed by 25 μL/well of the pre-mixed cytokine/chemokinebeads to all wells and the plate incubated overnight on a plate shakerat 4° C. The following day, standards, controls and sample volumes wereremoved by vacuum filtration and washed two times with 200 μL/well washbuffer and 25 μL/well biotinylated detection antibodies added to allwells and incubated on a plate shaker for 1 hr at RT. The reaction wasdeveloped by adding 25 μL/well of streptavidin-phycoerythrin to allwells and incubated for a further 30 min at RT on a plate shaker. Theplate was then washed twice with assay buffer and a final volume of 1504of sheath fluid added to all wells and beads re-suspended. The plate wasread using a Luminex 100™ IS instrument and software package (LuminexCorporation, Texas, USA) and the mean fluorescent intensity dataanalyzed using a weighted 5-parameter logistic method to yieldcytokine/chemokine concentrations (pg/mL) in the supernatants.

Results

The results shown in FIG. 3 indicate that treatment with L-sulforaphaneproduces a reduction in the chemokines and cytokines examined, with morepronounced effects than the classical histone deacetylase inhibitors,Trichostatin A (TSA), suberoyl anilide hydroxamic acid (SAHA), andsodium butyrate (NaB).

Example 3 L-Sulforaphane Prevents Allergic Airways Disease andNaphthalene-Induced Airway Epithelial Damage Summary

Administration of LSF prevented the damage and detrimental effects inmouse models of allergic airway reactions and chemical-induced airwayepithelial damage.

Methods

An established model of ovalbumin (OVA)-induced AAD was used aspreviously described (Temelkovski et al., 1998). This model includesmany of the pathological features of human asthma including increasedallergic responses indicated by increased immunoglobulin E against OVA(OVA-specific IgE), epithelial remodeling, goblet cell metaplasia,subepithelial collagen deposition (fibrosis) and airwayhyperresponsiveness. Briefly, 6-8 week old mice (Balb/c) were sensitizedwith 10 μg of grade V OVA (Sigma Chemical, St Louis, Mo., USA) and 1 mgof aluminum potassium sulfate adjuvant (alum) in 500 μl salineintraperitoneally on day 0 and 14 and then challenged with nebulized2.5% (w/v) OVA in saline three days per week for six weeks to establishAAD. Ovalbumin-exposed mice were treated with 5 mg/kg L-sulforaphane(OVA-LSF, n=5) or vehicle control (OVA-VEH, n=15) intraperitoneallyfollowing each OVA nebulization (3 days per week for 6 weeks). A thirdgroup of mice, sensitized with saline/alum on days 0 and 14 andnebulized with saline 3 days per week for 6 weeks (n=15), served asadditional controls. All experimental procedures were approved by theInstitutional Animal Ethics Committee and followed the AustralianGuidelines for the Care and Use of Laboratory Animals for ScientificPurposes.

Morphometric analysis was performed on H&E stained lung tissue sections.Images of lung tissue sections were captured using a Digital camera (QImaging, Burnaby, British Columbia, Canada). A minimum of five bronchimeasuring 150-350 μm luminal diameter were analyzed per mouse usingImage Pro-Discovery software (Media Cybernetics, Silver Spring, Md.),which was calibrated with a reference micrometer slide. The thickness ofthe bronchial epithelial layer was measured by tracing around thebasement membrane and the luminal surface of epithelial cells using adigitizer (Aiptek, Irvine, Calif.) and calculating the mean distancebetween the lines by Image Pro-Discovery software (Media Cybernetics).

For immunofluorescence, tissue sections were blocked for 1 hour usingSuperblock (Thermo Scientific) at room temperature followed by a 5minute wash using 0.5% Tween 20, 0.1% Triton X-100 in phosphate bufferedsaline (PBS-TT). Tissues were exposed to primary monoclonal antibodiesanti-Annexin V (rabbit; Epitomics) and anti-histone deacetylase 8(mouse, Sigma), diluted in 1% BSA (1:500). Primary antibodies wereincubated in a dark humidified chamber overnight. Following three 10minute washes in PBS-TT, tissues were incubated with secondaryantibodies, goat anti-mouse Alexa 488 (Molecular Probes) and goatanti-rabbit 546 (Molecular Probes) diluted in 1% BSA (1:500) in a darkhumidified chamber for one hour on a rotating platform. Following three10 minute washes in PBS-TT, tissues were mounted using Prolong GoldAntifade with DAPI (Invitrogen Molecular Probes). Slides were incubatedovernight at 4° C. before imaging. Images were acquired using an OlympusBX61 fluorescence microscope automated with FVII Camera.

For studies using naphthalene-induced airway epithelial damage byL-sulforaphane, female wild type (C57B6J) mice between the ages of sixto eight weeks were injected with naphthalene (200 mg/kg)intraperitoneally (ip) or with corn oil (vehicle control, volumes werenormalized for body weight). Mice were monitored for up to 72 hours (thepoint by which re-epithelialization has occurred) and mice were culledat 24 and 72 hours for analysis. The treatment groups received an ipinjection of 5 mg/kg L-sulforaphane (LSF) or 1 mg/kg dexamethasone(DEX). Histological analysis was performed on hematoxylin and eosin orMason's trichrome stained lung sections.

Results

Histological examination indicates that L-sulforaphane has beneficialeffects compared to ovalbumin-sensitized mice with reductions in gobletcell hyperplasia, inflammation and airway wall thickness being observed(FIG. 4A-C). Mean airway wall thickness and epithelial thickness werequantitated by morphometric analysis (FIG. 5). Strong staining ofAnnexin V was found largely in bronchial epithelium and peribronchialinflammatory cells in mice treated with OVA-VEH. Weak Annexin V stainingwas present in mice treated with OVA-LSF indicating a reduction inapoptosis. In contrast, Annexin V staining was not observed in theepithelium in saline control mice (FIG. 4D-F).

Histological examination indicates that L-sulforaphane reducesepithelial denudation at 24 hours to a level which analogous to thatobserved with the glucocorticoid dexamethasone (FIG. 6). Mason'strichrome staining indicates that L-sulforaphane reduces laminareticularis thickness at 72 hours with efficacy similar to that ofdexamethasone (FIG. 7).

Example 4 Protection of Cardiac Myocytes by Doxorubicin-Induced DNADamage by L-Sulforaphane Using DNA Double-Strand Breaks as a Model(γH2AX Foci) Methods

Rat embryonic ventricular myocardial H9c2 cells were obtained from theAmerican Type Culture Collection and were grown as monolayers inDulbecco's modified Eagle's medium (DMEM), containing 10% fetal bovineserum (FBS, In Vitro Technologies, Victoria, Australia), 100 U//mlpenicillin and 100 μg/ml streptomycin (Invitrogen, Carlsbad, Calif.,US), at 37° C. in a humidified atmosphere with 5% CO₂. Prior toconfluence (typically 60-70%), cells were passaged using 0.5%trypsin-EDTA (Invitrogen) and centrifugation (250×g for 5 minutes) andseeded at ratios of 1:2 or 1:3 in DMEM containing 10% FBS for 24 or 48hours. Cells were then cultured in DMEM containing 10 nMall-trans-retinoic acid (Sigma-Aldrich, St. Luis, Mo., US) for 7 daysand the culture media was changed daily to obtain cardiac myocytes.Cells were incubated with 1 μM doxorubicin for 1 hour, washed twice withphosphate buffered saline without calcium and magnesium and wereincubated for a further 24 hours in fresh media. To examine the effectsof L-sulforaphane, H9c2 cells were pre-treated with 0, 10, 15 and 30 μMfor 24 hours prior to treatment with doxorubicin. The number of γH2AXfoci in H9c2 cell nuclei were quantitated as described previously (Mahet al., 2010).

Results

Representative immunofluorescence microscopy images of H9c2 cells werepre-treated with 0, 10, 15 and 30 μM for 24 hours prior to treatmentwith doxorubicin are shown in FIG. 8. The results were quantified, andare presented in FIG. 9. The findings indicate that L-sulforaphane (LSF)attenuates doxorubicin-induced accumulation of γH2AX foci in H9c2 cellsindicating potent antioxidant effects.

Example 5 Downregulation of Innate Immune and Inflammatory Pathways inPBMC by L-Sulforaphane Summary

Horse PBMC were treated LSF or control in triplicate were analysed withmRNAseq. The sequencing run generated 161 million sequence tags thatwere used to measure the abundance of transcripts. Over 5,000differentially regulated genes were identified. Pathways related tointerferon signalling, STAT1/2 targets and autoimmune/auto-inflammatorydiseases were strongly downregulated, suggesting that LSF has apotential to be a potent anti-inflammatory therapeutic. The expressionof CD markers, immunoglobulin containing genes and interleukins are alsostrongly down-regulated.

Methods

RNA Isolation

RNA was isolated from trizol homogenates using the recommended organicphase separation technique followed by precipitation with isopropanoland resuspension in RNase free water. RNA was analysed on the MultiNAbioanalyzer (Shimadzu).

mRNA Sequencing

NEBNext® Poly(A) mRNA Magnetic Isolation Module was used to enrich mRNAfrom 1 μg of total RNA. We used the NEBNext® Ultra™ Directional RNALibrary Prep Kit for Illumina® to generate barcoded libraries. Librarieswere validated on the MultiNA bioanalyzer (Shimadzu) and pooled toequimolar ratios for sequencing. The pooled library was sequenced at theAustralian Genome Research Facility (Melbourne) on Illumina HiSeq2500instrument with version 4 single end flow cell for 60 cycles.

Bioinformatics Analysis

Data Processing and Technical Quality Control

Sequence data underwent quality trimming to remove low quality basesfrom the 3′ end of reads using FASTXToolkit (version 0.0.14) using aPhred quality threshold of 20 and minimum 20 nt read length. STARversion 3.2.0.1 [PMID:23104886] was used to align reads to the Horsegenome (Equus_caballus.EquCab2.dna.toplevel.fa) downloaded from Ensembl.We used Ensembl version 77 gene annotations(Equus_caballus.EquCab2.77.gtf). Exonmapped reads were counted usingfeatureCounts version 1.4.2 [PMID: 24227677]. Genes with fewer than 10reads per sample on average were excluded from downstream analysis.Statistical analysis of differential gene expression was conducted usingedgeR software version 0.20 with the default settings [PMID: 19910308].To facilitate pathway analysis, horse gene identifiers were mapped tohuman gene names using horse-human homolog relationship table downloadedfrom Ensembl BioMart. Pathway analysis was performed using GSEAPsoftware version gsea22.1.0 using the unweighted “classic” scoringscheme. Gene sets for pathway analysis were downloaded from MSigDB.ENCODE and Mouse ENCODE transcription factor binding site (TFBS) datawere mined to generate gene sets of transcription factor targets thatwere also queried using GSEAP as described in the supplementarymaterial. False discovery rate (FDR) adjusted p-values ≦0.05 wereconsidered significant.

Results

Nearly all reads (99.93%) passed QC filtering and 83.5% of reads couldbe uniquely aligned. Alignment statistics are shown in Table 2.

TABLE 2 Alignment set statistics. Sample Ctrl1 Ctrl2 Ctrl3 LSF1 LSF2LSF3 Total reads 23184730 24378587 31178787 24141494 29984514 28352005QC passed reads 23170012 24362373 31156618 24125584 29962232 28332079Average input read length 59 59 59 59 59 59 Uniquely mapped reads number19186428 19603100 25257061 20424251 25531078 24544230 Uniquely mappedreads % 82.82% 80.45% 81.07% 84.64% 85.22% 86.63% Average mapped length59.3 59.3 59.3 59.3 59.3 59.3 Number of splices: Total 1532824 14954901878671 1472027 1669309 1947980 Number of splices: GT/AG 1516298 14795001858339 1455716 1650330 1926500 Number of splices: GC/AG 10147 986312591 10076 11347 13298 Number of splices: AT/AC 867 788 994 775 9171097 Number of splices: Non-canonical 5512 5339 6747 5460 6715 7085Mismatch rate per base, % 0.31% 0.38% 0.32% 0.36% 0.29% 0.35% Deletionrate per base 0.01% 0.01% 0.01% 0.01% 0.01% 0.01% Deletion averagelength 2.5 2.48 2.48 2.54 2.56 2.56 Insertion rate per base 0.01% 0.01%0.01% 0.01% 0.01% 0.01% Insertion average length 2.5 2.53 2.51 2.52 2.542.51 Number of reads mapped 2990229 3670724 4401558 2718150 31416402673452 to multiple loci % of reads mapped to multiple loci 12.91%15.06% 14.13% 11.27% 10.50% 9.43% Number of reads mapped to 25775 2643034890 30008 39826 32422 too many loci % of reads mapped to too many loci0.11% 0.11% 0.11% 0.12% 0.13% 0.11% Assigned 8073651 8046910 101590137874621 9115250 10248009 Unassigned_Ambiguity 10280 10496 13343 915610743 12572 Unassigned_MultiMapping 0 0 0 0 0 0 Unassigned_NoFeatures11102497 11545694 15084705 12540474 16405085 14283649Unassigned_Unmapped 0 0 0 0 0 0 Unassigned_MappingQuality 1134290314213425 16314170 9907186 11215053 9364434 Unassigned_FragementLength 00 0 0 0 0 Unassigned_Chimera 0 0 0 0 0 0

After exclusion of genes below the detection threshold (average of 10reads per sample), transcripts from 10,800 ensembl genes were detected.Differential expression analysis of three control samples versus threeLSF treated samples resulted in 5951 differentially expressed genes. Ofthese, 2939 were up-regulated and 3012 were down-regulated. Table 3 showthe top 20 up and down-regulated genes. CPM is the counts per million,and is a measure of baseline expression level

TABLE 3 Top differnetially regulated genes. Ensembl Accession GeneNameLog2 Fold Change Log2 CPM Adj P-value ENSECAG00000022834 WDR59 2.19 7.472.50E−187 ENSECAG00000020307 DENND2A 3.67 5.67 2.30E−186ENSECAG00000021598 ZFAND2A 2.22 6.93 1.30E−182 ENSECAG00000015982 TXNRD12.13 9.70 3.80E−159 ENSECAG00000014974 GSAP 2.00 7.78 2.80E−149ENSECAG00000022696 SLC7A11 2.34 9.33 3.60E−137 ENSECAG00000021447 ASPH1.93 7.46 1.60E−132 ENSECAG00000015342 IL8 3.54 9.79 4.70E−111ENSECAG00000024408 STAC2 3.38 4.53 5.90E−102 ENSECAG00000000362 STXBP51.57 8.42 3.30E−101 ENSECAG00000023011 RIPK2 2.01 6.96 2.50E−097ENSECAG00000014513 TNFSF15 2.40 4.87 3.00E−094 ENSECAG00000011797 MN-SOD1.93 9.62 6.20E−093 ENSECAG00000003192 FTL 1.51 9.74 1.10E−092ENSECAG00000024482 ME1 1.47 7.54 2.10E−092 ENSECAG00000013324 TXN 1.8711.06 2.50E−091 ENSECAG00000014514 TUBA4A 1.30 8.38 5.80E−088ENSECAG00000013560 OSGIN2 1.88 6.08 1.70E−086 ENSECAG00000007922 KCNJ22.06 6.82 3.70E−086 ENSECAG00000008693 PTPN12 1.43 8.67 7.40E−081ENSECAG00000024705 MAFB −2.66 7.82 2.47E−248 ENSECAG00000003015 SERPINB2−4.65 9.48 4.40E−238 ENSECAG00000013723 SLC7A8 −3.35 6.47 5.00E−230ENSECAG00000017398 CD180 −2.97 8.21 1.00E−223 ENSECAG00000010251 SLC37A2−2.72 6.53 2.30E−215 ENSECAG00000011733 RNASE6 −2.45 7.49 2.20E−214ENSECAG00000020052 TNS3 −3.09 6.65 1.30E−213 ENSECAG00000014707 CXCL9−2.55 8.90 2.50E−213 ENSECAG00000019442 CYP1B1 −3.83 5.51 3.50E−203ENSECAG00000013457 CSF3R −3.76 6.65 2.90E−202 ENSECAG00000023720 SLAMF8−2.75 6.58 2.10E−193 ENSECAG00000000946 CMKLR1 −3.79 5.31 5.70E−174ENSECAG00000015726 NR1H3 −2.56 7.97 1.30E−173 ENSECAG00000004709 C2−2.13 6.95 1.90E−164 ENSECAG00000008852 ABCA1 −4.37 9.36 1.00E−159ENSECAG00000000153 FRMPD4 −3.80 5.35 1.00E−156 ENSECAG00000023046 SORCS1−2.71 6.36 9.60E−155 ENSECAG00000022371 APOBEC3Z1B −3.67 5.64 4.00E−153ENSECAG00000006290 TGM2 −3.99 4.77 3.60E−151 ENSECAG00000024841 PLEKHA4−3.60 4.72 5.40E−151Gene expression data are visualised by multidimensional scaling (MDS)plot (FIG. 10), which displays the variability of the samples asdistance on a two-dimensional plot. The plot shows separation of thesamples groups on dimension 1 (x axis), indicating that the treatment isthe major source of variability in the experiment. On the seconddimension (y axis), the LSF treated samples show some variability,indicating technical/biological variability is the second source ofvariation in the experiment. The smearplot shown in FIG. 11 allowsexamination of relationship of overall baseline expression (Log CPM, xaxis) with the fold change (Log FC, y axis).

Next pathways analysis using Gene Set Enrichment Analysis (GSEA) inthree stages was performed: (1) Canonical pathways curated by REACTOME;(2) MSigDB gene sets; and (3) ENCODE TFBS. These help to understand thebroad trends in major pathways, the specific similarities to previousprofiling experiments and the chromatin level regulation mediated bytranscription factors. NES in Table 4 is the normalised enrichment scorederived by GSEA and is a measure of how strong an up- or down-regulationtrend is.

TABLE 4 Pathway analysis of bleomycin treatment with Reactome gene sets.GENE SET NES FDR q-val GENERIC TRANSCRIPTION PATHWAY 4.23 0 MRNAPROCESSING 3.68 0 PROCESSING OF CAPPED INTRON CONTAINING PRE MRNA 3.61 0MRNA 3 END PROCESSING 3.5 0 CLEAVAGE OF GROWING TRANSCRIPT IN THETERMINATION REGION 3.33 0 RNA POL II TRANSCRIPTION 3.32 0 TRANSCRIPTION3.25 0 DOWNSTREAM SIGNALING EVENTS OF B CELL RECEPTOR BCR 3.12 0 MRNASPLICING 3.09 0 TRANSPORT OF MATURE TRANSCRIPT TO CYTOPLASM 3.02 0ANTIGEN PROCESSING UBIQUITINATION PROTEASOME DEGRADATION 2.7 0.001ASSOCIATION OF TRIC CCT WITH TARGET PROTEINS DURING BIOSYNTHESIS 2.620.001 REGULATION OF MRNA STABILITY BY PROTEINS THAT BIND AU RICHELEMENTS 2.6 0.002 RECRUITMENT OF MITOTIC CENTROSOME PROTEINS ANDCOMPLEXES 2.55 0.002 PI3K EVENTS IN ERBB4 SIGNALING 2.52 0.002 PI3KEVENTS IN ERBB2 SIGNALING 2.49 0.003 PIP3 ACTIVATES AKT SIGNALING 2.480.002 REGULATION OF ORNITHINE DECARBOXYLASE ODC 2.45 0.003 LATE PHASE OFHIV LIFE CYCLE 2.44 0.003 HIV LIFE CYCLE 2.41 0.004 INTERFERON ALPHABETA SIGNALING −4 0 INTERFERON GAMMA SIGNALING −3.9 0 GPCR DOWNSTREAMSIGNALING −3.63 0 IMMUNOREGULATORY INTERACTIONS BETWEEN A LYMPHOID AND ANON −3.37 0 LYMPHOID CELL SIGNALING BY GPCR −3.35 0 INTERFERON SIGNALING−3.18 0 SIGNALING BY RHO GTPASES −2.87 0 CYTOKINE SIGNALING IN IMMUNESYSTEM −2.84 0 G ALPHA S SIGNALLING EVENTS −2.82 0 TCA CYCLE ANDRESPIRATORY ELECTRON TRANSPORT −2.8 0 RESPIRATORY ELECTRON TRANSPORT ATPSYNTHESIS BY CHEMIOSMOTIC −2.69 0 COUPLING AND HEAT PRODUCTION BYUNCOUPLING PROTEINS CLASS A1 RHODOPSIN LIKE RECEPTORS −2.68 0 GPCRLIGAND BINDING −2.67 0.001 ACTIVATION OF KAINATE RECEPTORS UPONGLUTAMATE BINDING −2.56 0.001 OPIOID SIGNALLING −2.55 0.001 G ALPHA1213SIGNALLING EVENTS −2.54 0.001 MHC CLASS II ANTIGEN PRESENTATION −2.470.002 PEPTIDE CHAIN ELONGATION −2.43 0.003 G ALPHA I SIGNALLING EVENTS−2.41 0.003 GABA RECEPTOR ACTIVATION −2.39 0.004The pathway analysis shows strong downregulation of type I (alpha/beta)and type II (gamma) interferon (IFN) signaling, which is an indicationof a broad downregulation of innate immune response, that is normallyassociated with inflammation. These are shown in detail in FIG. 12. GSEAwith the larger MSigDB gene set library (Table 5) identified manyassociations background information on these gene sets, and can be foundat the Broad institute website. The MYST2 and lupusrelated gene sets areshown in FIG. 13. GSEA with ENCODE TF target gene sets identifiedupregulation of targets of general transcription factors (POL2, TAF1)and downregulation of STAT1, STAT2 and RAD21 targets. The STAT1 andSTAT2 target gene sets are shown in more detail in FIG. 14.

TABLE 5 MSigDB GSEA analysis. GS DETAILS NES FDR q-val HAMAI APOPTOSISVIA TRAIL UP 5.42 0 SHEN SMARCA2 TARGETS UP 5.39 0 GCM MYST2 4.99 0 GCMZNF198 4.95 0 MILI PSEUDOPODIA HAPTOTAXIS UP 4.81 0 GCM UBE2N 4.65 0 GCMDFFA 4.6 0 CHEN HOXA5 TARGETS 9HR UP 4.3 0 GCM SUFU 4.3 0 GSE 10239NAIVE VS MEMORY CD8 TCELL UP 4.2 0 MRNA METABOLIC PROCESS 4.19 0ATGTTAA, MIR-302C 4.19 0 REACTOME GENERIC TRANSCRIPTION PATHWAY 4.19 0GSE29617 CTRL VS DAY3 TIV FLU VACCINE PBMC 2008 UP 4.12 0 CTTGTAT,MIR-381 4.1 0 GCM MLL 4.08 0 RNA PROCESSING 4.02 0 V$NFMUE1 Q6 3.98 0GABRIELY MIR21 TARGETS 3.8 0 GCM RAB10 3.8 0 MODULE 84 −7.87 0 GSE10325LUPUS CD4 TCELL VS LUPUS MYELOID DN −7.15 0 GSE13485 CTRL VS DAY7 YF17DVACCINE PBMC DN −6.96 0 GSE10325 LUPUS BCELL VS LUPUS MYELOID DN −6.47 0GSE10325 CD4 TCELL VS MYELOID DN −6.35 0 MODULE 46 −6.22 0 GSE24634 TREGVS TCONV POST DAY10 IL4 CONVERSION DN −5.97 0 GSE29618 BCELL VS MDC DAY7FLU VACCINE DN −5.93 0 MODULE 45 −5.9 0 MODULE 75 −5.9 0 GSE24634 IL4 VSCTRL TREATED NAIVE TCELL DAY5 UP −5.8 0 MCLACHLAN DENTAL CARIES UP −5.670 WALLACE PROSTATE CANCER RACE UP −5.66 0 GSE13485 CTRL VS DAY3 YF17DVACCINE PBMC DN −5.66 0 GSE11057 CD4 EFF MEM VS PBMC DN −5.63 0 GSE13485DAY3 VS DAY7 YF17D VACCINE PBMC DN −5.63 0 GSE13485 DAY7 VS DAY21 YF17DVACCINE PBMC UP −5.61 0 GSE22886 NAIVE CD4 TCELL VS MONOCYTE DN −5.6 0MEISSNER BRAIN HCP WITH H3K4ME3 AND H3K27ME3 −5.6 0 FULCHER INFLAMMATORYRESPONSE LECTIN VS LPS DN −5.59 0

TABLE 6 Pathway analysis using ENCODE TF binding targets. GS DETAILS NESFDR q-val H1HESC POL2 3.65 0 HELAS3_TAF1 3.48 0 K562_HEY1 3.48 0GM12878_ETS1 3.47 0 H1HESC_POL2 3.42 0 GM12878_YY1 3.41 0 HEPG2_ZBTB333.35 0 H1HESC_POL2 3.34 0 H1HESC_POL2 3.33 0 HEPG2_ZBTB33 3.23 0SHSY5Y_GATA3 3.13 0 K582_TR4UCD 3.13 0 GM12878_ZBTB33 3.05 0 K562_KAP13.04 0 HEPG2_GRP20_FORSKLN 3.01 0 SKNMC_POL2 2.91 0 GM12878_ZBTB33 2.890 H1NEURONS_NRSF 2.84 0 U2OS_SETDB1 2.83 0 K562_ZBTB33 2.78 0K562_STAT1_IFNA6H −4.54 0 K562_STAT2_IFNA6H −4.45 0 GM12878RAD21 −3.95 0GM12878_RAD21 −3.79 0 MCF10AES_STAT3 −3.75 0 K562_STAT1_IFNA30 −3.71 0GM12878_EBF_SC137065 −3.67 0 HELAS3_RAD21 −3.63 0 SKNSH_SMC3 −3.63 0ECC1_RAD21 −3.56 0 GM12878_PU1 −3.58 0 SKNSH_NFIC −3.5 0 ECC1_RAD21−3.45 0 ECC1_ERALPHA −3.45 0 HEPG2_RAD21 −3.44 0 GM12878_NFKB_TNFA −3.40 GM12878_PU1 −3.4 0 K562_STAT2_IFNA30 −3.39 0 SKNSH_TCF12 −3.34 0MCF7_RAD21 −3.31 0Based on the results showing broad downregulation by FSF of innateimmune responses normally associated with inflammation, furtherexamination was conducted of specific genes related to inflammatorysignalling such as cytokines, interleukins and their receptors. Threemajor groups of genese were focused on: (1) CD markers (FIG. 15A), (2)immunoglobulins (FIG. 15B), and (3) endogenous ligands (FIG. 15C).

Of the 161 million sequence tags that were used to measure the abundanceof transcripts, over 5,000 differentially regulated genes wereidentified. Pathways related to interferon signalling, STAT1/2 targetsand autoimmune/auto-inflammatory diseases were strongly downregulated,suggesting that LSF has a potential to be a potent anti-inflammatorytherapeutic that may be useful to treat a range of diseases that areassociated with systemic or local inflammation. The expression of CDmarkers, immunoglobulin containing genes and interleukins are alsostrongly down-regulated.

The inventions being thus described, it will be obvious that the samemay be varied in many ways. Such variations are not to be regarded as adeparture from the spirit and scope of the inventions.

We claim:
 1. A pharmaceutical composition for treatment, suppressionand/or amelioration of a condition or disease associated withinflammation or oxidative stress, wherein said composition comprisesL-sulforaphane (LSF), an LSF derived and/or substituted compound, and/oran LSF analogue.
 2. The composition of claim 1 wherein the LSF, LSFderived and/or substituted compound, and/or an LSF analogue is isolated,purified and/or synthesized and has the following formula:

and has an inhibitory effect against one or more HDAC proteins.
 3. Thecomposition of claim 1 wherein R is selected from the group consistingof hydrogen, substituted or unsubstituted alkyl, substituted orunsubstituted alkenyl, substituted or unsubstituted alkynyl, substitutedor unsubstituted aryl, substituted or unsubstituted heterocyclyl,substituted or unsubstituted acyl, ORa, SRa, SORa, SO2Ra, OSO2Ra,OSO3Ra, NO2, NHRa, N(Ra)2, ═N—Ra, N(Ra)CORa, N(CORa)2, N(Ra)SO2R′,N(Ra)C(═NRa)N(Ra)Ra, CN, halogen, CORa, COORa, OCORa, OCOORa, OCONHRa,OCON(Ra)2, CONHRa, CON(Ra)2, CON(Ra)ORa, CON(Ra)SO2Ra, PO(ORa)2,PO(ORa)Ra, PO(ORa)(N(Ra)Ra) and aminoacid ester having inhibitoryefficacy against the LSD1 protein; and further wherein each of the Ragroups is independently selected from the group consisting of hydrogen,substituted or unsubstituted alkyl, substituted or unsubstitutedalkenyl, substituted or unsubstituted alkynyl, substituted orunsubstituted aryl, and substituted or unsubstituted heterocyclyl,substituted or unsubstituted acyl, and the like; and further whereineach of the substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl,heterocyclyl, and/or acyl groups are C1-28 (including all rangestherein).
 4. The composition of claim 3 wherein the LSF, LSF derivedand/or substituted compound, and/or an LSF analogue has the followingformula:


5. The composition of claim 1 wherein said condition or disease ispulmonary edema or exercise-induced pulmonary hemorrhage (EIPH).
 6. Thecomposition of claim 1 further comprising one or more of hydroxytyrosol,oleuropein, N-acetylcysteine, L-proline, glycine, and taurine.
 7. Amethod of treating or preventing a condition or disease associated withinflammation or oxidative stress, comprising administering to a subjectin need thereof a composition comprising L-sulforaphane (LSF), an LSFderived and/or substituted compound, and/or an LSF analogue.
 8. Themethod of claim 7 wherein the LSF, LSF derived and/or substitutedcompound, and/or an LSF analogue is isolated, purified and/orsynthesized and has the following formula:

and has an inhibitory effect against one or more HDAC proteins.
 9. Themethod of claim 7 wherein R is selected from the group consisting ofhydrogen, substituted or unsubstituted alkyl, substituted orunsubstituted alkenyl, substituted or unsubstituted alkynyl, substitutedor unsubstituted aryl, substituted or unsubstituted heterocyclyl,substituted or unsubstituted acyl, ORa, SRa, SORa, SO2Ra, OSO2Ra,OSO3Ra, NO2, NHRa, N(Ra)2, ═N—Ra, N(Ra)CORa, N(CORa)2, N(Ra)SO2R′,N(Ra)C(═NRa)N(Ra)Ra, CN, halogen, CORa, COORa, OCORa, OCOORa, OCONHRa,OCON(Ra)2, CONHRa, CON(Ra)2, CON(Ra)ORa, CON(Ra)SO2Ra, PO(ORa)2,PO(ORa)Ra, PO(ORa)(N(Ra)Ra) and aminoacid ester having inhibitoryefficacy against the LSD1 protein; and further wherein each of the Ragroups is independently selected from the group consisting of hydrogen,substituted or unsubstituted alkyl, substituted or unsubstitutedalkenyl, substituted or unsubstituted alkynyl, substituted orunsubstituted aryl, and substituted or unsubstituted heterocyclyl,substituted or unsubstituted acyl, and the like; and further whereineach of the substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl,heterocyclyl, and/or acyl groups are C1-28 (including all rangestherein).
 10. The method of claim 7 wherein the LSF, LSF derived and/orsubstituted compound, and/or an LSF analogue has the following formula:


11. The method of claim 7 further comprising administering to saidindividual one or more of hydroxytyrosol, oleuropein, N-acetylcysteine,L-proline, glycine, and taurine.
 12. The method of claim 7 wherein saiddisease or condition is pulmonary edema or exercise-induced pulmonaryhemorrhage (EIPH).
 13. A method for increasing or decreasing geneexpression in a cell comprising contacting said cell with a compositioncomprising L-sulforaphane (LSF), an LSF derived and/or substitutedcompound, and/or an LSF analogue.
 14. The method of claim 13 furthercomprising inhibiting one or more histone deacetylases (HDAC) in saidcell.
 15. The method of claim 14 wherein said HDAC is HDAC8.
 16. Themethod of claim 14 further comprising administering comprisingcontacting said cell with one or more of hydroxytyrosol, oleuropein,N-acetylcysteine, L-proline, glycine, and taurine.
 17. The method ofclaim 13 further comprising increasing lysine acetylation of a histonepolypeptide in said cell.
 18. The method of claim 13 wherein saidincrease of decrease of gene expression improves cell viability.
 19. Amethod for treating or preventing oxidative stress in an individual orcell comprising contacting said individual or cell with a with acomposition comprising L-sulforaphane (LSF), an LSF derived and/orsubstituted compound, and/or an LSF analogue.
 20. The method of claim 19wherein the LSF, LSF derived and/or substituted compound, and/or an LSFanalogue is isolated, purified and/or synthesized and has the followingformula:

and has an inhibitory effect against one or more HDAC proteins.